The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression and interaction. Transgenics technology has also been used to produce models for various diseases in humans and other animals and is among the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. From an economic perspective, the use of transgenic technology to convert animals into “protein factories” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123) offers significant advantages over more conventional methods of protein production by gene expression.
One system useful for expressing foreign proteins is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct, which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk. In the past exogenous protein production has been performed in the avian reproductive system specifically targeting the avian oviduct.
Advantages of targeting the avian oviduct for exogenous protein expression can include proper folding and post-translation modification of the target protein, the ease of product recovery, and a shorter developmental period of birds such as chickens compared to other animal species.
Directing expression of a heterologous gene product in the oviduct of a transgenic avian can be significantly advantageous over ubiquitous expression in the bird. That is, the consequences of ubiquitous expression of a bioactive gene product in a host animal may be undesirable. For example, in certain instances the ubiquitous presence of the recombinant protein may be harmful to the development of the avian which can kill the bird. Additionally, the bird's health may be negatively effected leading to reduced levels of protein production.
By weight, approximately 60% of an avian egg is composed of albumen which is composed of four major protein components; ovalbumin, ovomucoid, lysozyme and ovotransferrin with ovalbumin and ovomucoid being present in the greatest quantities.
The ovalbumin promoter, ovomucoid promoter and lysozyme promoter have been successfully employed for the production of heterologous (exogenous) protein in the oviduct of transgenic avians in the past. See, for example, U.S. Pat. No. 6,875,588, issued Apr. 5, 2005; U.S. Pat. No. 7,176,300, issued Feb. 13, 2007; U.S. Pat. No. 7,199,279, issued Apr. 3, 2007; and US patent publication No. 2006/0130170, published Jun. 15, 2006 (the disclosures of each of these three issued patents and one published patent application are incorporated in their entirety herein by reference) which discloses the production of exogenous protein in the avian oviduct facilitated by various avian promoters which are primarily or exclusively expressed in the oviduct. Though expression levels in avians using the promoters and fragments of the promoters disclosed in these issued patents and published application have been at useful levels, the yields have typically been well below 0.1 mg/ml of egg white.
What is needed is a system that will provide for high level expression of an exogenous coding sequence in the cells of a transgenic avian, in particular, in the oviduct cells (e.g., tubular gland cells) of a transgenic avian.